2,4-Diamino-6-(hydroxymethyl)pyrido[2,3-d]pyrimidine

ABSTRACT

There is disclosed a novel intermediate useful in the preparation of pyrido [2,3-d] pyrimidines, which includes N-[4-[(2-amino-4(3H)-oxopyrido[2,3-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glutamic acid (5-deazafolic acid), N-[4-[[2-amino-4(3H)-oxopyrido[2,3-d]-pyrimidin-6-yl)methyl]methylamino]benzoyl]-L-glutamic acid (5-deaza-N 10  -methylfolic acid), N-(4-[(2,4-diaminopyrido[2,3-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glutamic acid (5-deazaaminopterin), and N-[4-[[2,4-diaminopyrido[2,3-d]pyrimidin-6-yl)methyl]methylamino]benzoyl]-L-glutamic acid (5-deazamethotrexate). This intermediate is the compound 2,4-diamino-6-(hydroxymethyl)pyrido-[2,3-d]pyrimidine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of our copending applicationSer. No. 338,542, filed Jan. 11, 1982, now U.S. Pat. No. 4,431,805;which is a continuation-in-part of our application Ser. No. 305,907,filed Sept. 25, 1981, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to intermediates useful in the preparation ofpyrido[2,3-]pyrimidines, which includesN-[4-[(2-amino-4(3H)-oxopyrido[2,3-d]-pyrimidin-6-yl)methylamino]benzoyl]-L-glutamicacid (5-deazafolic acid),N-[4-[[(2-amino-4(3H)-oxopyrido[2,3-d]-pyrimidin-6-yl)methyl]methylamino]benzoyl]-L-glutamicacid (5-deaza-N¹⁰ -methylfolic acid),N-[4-[(2,4-diaminopyrido[2,3-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glutamicacid (5-deazaaminopterin), andN-[4-[[(2,4-diaminopyrido[2,3-d]pyrimidin-6-yl)methyl]methylamino]benzoyl]-L-glutamicacid (5-deazamethotrexate). This invention also relates to processes forusing said intermediates; to the novel compounds 5-deaza-N¹⁰-substituted aminopterin and 5-deaza-N¹⁰ -substituted folic acid; and tomethods for preparing such novel compounds.

Powerful dihydrofolate reductase inhibitors such as aminopterin andmethotrexate are known folic acid antagonists useful in the suppressionand treatment of acute leukemia and related conditions. They have astheir principal mechanism of action a competitive inhibition of theenzyme dihydrofolate reductase. Folic acid and its 7,8-dihydroderivative must be reduced to tetrahydrofolic acid by this enzyme in theprocess of DNA synthesis and cellular reproduction. Compounds havingantifolate activity such as aminopterin and methotrexate inhibit thereduction of both folic acid and 7,8-dihydrofolic acid and interferewith tissue-cell reproduction.

Several types of quinazolinyl (5,8-dideazapteridinyl) analogs of folicacid, aminopterin, and methotrexate were reported to be inhibitors bothof dihydrofolate reductase and thymidylate synthetase [A. H. Calvert, T.R. Jones, P. J. Dady, B. Grzelakowska, R. M. Paine, G. A. Taylor and K.R. Harrap, Europ. J. Cancer, 16, 713 (1980); K. J. Scanlon, B. A.Moroson, J. R. Bertino and J. B. Hynes, Mol. Pharmacol., 16, 261 (1979);O. D. Bird, J. W. Vaitkus and J. Clarke, Mol. Pharmacol., 6, 573(1970)]. Recently,N-[4-[N-[(2-amino-4-hydroxy-6-quinazolinyl)methyl]prop-2-ynylamino]benzoyl]-L-glutamicacid (5,8-dideaza-10-propargylfolic acid) was identified as a potentinhibitor of thymidylate synthetase [T. R. Jones, A. H. Calvert, A. L.Jackman, S. J. Brown, M. Jones and K. R. Harrap, Europ. J. Cancer, 17,11 (1981)]. This enzyme catalyzes the de novo synthesis of thymidinenucleotides, which are required for DNA synthesis.

The synthesis of derivatives of the pyrido[2,3-d]pyrimidine ring systemhas been reviewed by W. J. Irwin and D. G. Wibberley, Advan. Heterocycl.Chem., 10, 149 (1969), which covers the literature to the beginning of1968. Although many methods are reported in this review, major routes tothis ring system include the cyclization of the functional derivativesof 2-aminonicotinic acids with various reagents [e.g., R. K. Robins andG. H. Hitchings, J. Am. Chem. Soc., 77, 2256 (1955)], and the reactionof derivatives of 4-aminopyrimidine with 1,3-dicarbonyl compounds ortheir masked derivatives [e.g., B. S. Hurlbert and B. F. Valenti, J.Med. Chem., 11, 708 (1968)]. The condensations of 4-aminopyrimidineswith malondialdehyde derivatives to give pyrido[2,3-d]pyrimidines arereported by R. Bernetti, F. Mancini and C. C. Price, J. Org. Chem., 27,2863 (1962), and B. S. Hurlbert and B. F. Valenti, J. Med. Chem., 11,708 (1968). A procedure for the preparation of5-oxo-(8H)-pyrido[2,3-d]pyrimidines was reported by B. H. Rizkalla andA. D. Broom, J. Org. Chem., 37, 3980 (1972). This reference disclosesthe following compound: ##STR1## The development of procedures for theconversion of the above compound toN-[4-[(2,4-diamino-5-oxo(8H)-pyrido[2,3-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glutamicacid (5-deaza-5-oxoaminopterin), i.e., a compound having the formula:##STR2## was reported by A. Srinivasan and A. D. Broom, J. Org. Chem.,45, 3746 (1980). In addition,N-[4-[(2-amino-4(3H)-oxo-10-formylpyrido[2,3-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glutamicacid (5-deaza-10-formylfolic acid), characterized only by spectral data,was reported to be formed from 5-deazafolate and formic acid by G. K.Smith, W. T. Mueller, P. A. Benkovic and S. J. Benkovic, Biochemistry,20, 1241 (1981). A method for preparing 5-deazafolate is not disclosed.

The inhibition of bacterial dihydrofolate reductase bypyrido[2,3-d]pyrimidines have been summarized in the Advan. Heterocycl.Chem. reference. Recently, a pyrido[2,3-d]pyrimidine derivative wasreported to be a potent lipid-soluble inhibitor of mammaliandihydrofolate reductase by E. M. Grivsky, S. Lee, C. W. Sigel, D. S.Duch and C. A. Nichol, J. Med. Chem., 23, 327 (1980). This referencediscloses the compound: ##STR3## Other derivatives of this ring systemhave been evaluated for antihyptertensive activity. Thus, L. R. Bennettet al, J. Med. Chem., 24, 382 (1981) reported that the followingcompound lowered blood pressure in the hypertensive rat: ##STR4##

The synthesis of 5-deazafolic acid has been reported by D. T. Hurst, "AnIntroduction to the Chemistry and Biochemistry of Pyrimidines, Purines,and Pteridines", John Wiley and Sons, Ltd., 231 (1980). The synthesis ofthis compound using as an intermediate2-amino-6-formyl-5-deazapteridine-4(3H-one, i.e., a compound having theformula: ##STR5## has been proposed by C. Temple, Jr. and J. A.Montgomery, "Synthesis of Potential Anticancer Agents", CancerChemotherapy National Service Center, Southern Research Institute,Progress Report 85, pages 1 and 2 (1966) and Progress Report 86, pages 8and 10 (1967). The synthesis of 5-deazafolic acid via a condensationreaction involving triformylmethane has been reported by C. P. Tseng,Dissertation Abstracts Int. B, 40, 3752 (1980). The thesis upon whichthis abstract was based, C. P. Tseng, Studies in Heterocyclic Chemistry,171-185 (1979) also describes unsuccessful work on the preparation of5-deaza-2,4-diamiopteridine-6-carboxaldehyde dimethyl acetal, i.e., acompound having the formula: the synthesis of 5-deaza-6-formylpterin;and the conversion of this compound to 5-deazafolic acid via acetylated5-deaza-6-formylpterin. The preparation of 5-deazaaminopterin via a longsequence of reactions involving the elaboration of a pyrimidineintermediate has been described by E. F. Elslager and J. Davoll,"Lectures in Heterocyclic Chemistry", 2, S-97, S-119-S-121 (1974).

SUMMARY OF THE INVENTION

The 5-deaza analogs of folic acid, N¹⁰ -substituted folic acid,aminopterin, N¹⁰ -substituted aminopterin and the diethyl ester ofaminopterin inhibit the growth of human epidermoid carcinoma cells No. 2and are active against leukemia in laboratory animals. The 5-deazaanalogs of folic acid and N¹⁰ -substituted folic acid referred to hereinhave the following structure: ##STR6## and the 5-deaza analogs ofaminopterin and N¹⁰ -substituted aminopterin referred to herein have thefollowing structure: ##STR7## wherein R is hydrogen, CH₃, CH₃ CH₂, CH₃CH₂ CH₂, CH₂ ═CHCH₂ or CH.tbd.CCH₂. Compounds of Formulas I and IIwherein R is other than hydrogen are novel compounds.

Novel intermediates have not been found which are useful in thepreparation of the compounds of Formulas I and II. These intermediateshave the structures: ##STR8## wherein Y is CHO, CH₂ OH or CH₂ Br; and Zis CH₂ OH or CH₂ Br.

Reductive alkylation and alkylation, respectively, of a dialkylp-aminobenzoyl-L-glutamate having the structure: ##STR9## with thecompounds of Formula III and Formula IIIA affords a compound having thestructure: ##STR10## wherein R is the same as previously defined and R₁is a lower alkyl group, i.e., a group containing up to six carbon atoms.Similarly, alkylation of a compound of Formula VI with a compound ofFormula IIIB affords a compound having the structure: ##STR11## whereinR and R₁ are the same as previously defined. It is preferred when R inthe compound of Formula VI is other than hydrogen, that the groups Y andZ on the compounds of Formulas III and IIIB be CH₂ Br.

The compounds of Formula VII and VIIA can be converted to the compoundsof Formulas II and I, respectively, by saponification. In addition, thecompounds of Formula II can be treated under more drastic conditionswith base to hydrolyze the 4-amino group according to the methoddescribed by R. Tratner, G. Elion, G. Hitchings, and D. Sharefkin, J.Org. Chem., 29, 2674 (1974) to give the compounds of Formula I. Also,the methylation of the compound of Formula II (R═H) with formaldehyde inthe presence of sodium cyanoborohydride provides the compound of FormulaII (R═CH₃). The compound of Formula I (R═CH₃) can also be prepared bymethylation of the compound of Formula I (R═H) with theformaldehyde-sodium cyanoborohydride combination.

DETAILED DESCRIPTION OF THE INVENTION

The compound of Formula III (Y═CHO) is prepared by reaction of acompound having the structure: ##STR12## with the quaternary salt oftriformylmethane (or its hydrolyzed derivatives) having the structure:

    CH[CH═N.sup.+ (CH.sub.3).sub.2 ].sub.3 3X.sup.-        V

wherein X is a halogen atom, preferably chlorine.

The synthesis of triformylmethane was reported by Z. Arnold and J.Zemlicka, Coll. Czech. Chem. Commun., 25, 1318 (1960) and Z. Arnold,Coll. Czech. Chem. Commun., 26, 3051 (1961). Thus, in one method,reaction of bromoacetic acid with the complex [(CH₃)₂ N═CHCl]⁺ Cl⁻resulting from treatment of N,N-dimethylformamide with phosphorusoxychloride gave a quaternary salt, probably V, which was treated withaqueous potassium carbonate to give triformylmethane. The isolation andpurification of the latter is difficult, and in the procedure describedherein, the intermediate quaternary salt or its hydrolyzed derivativesis used.

The condensation of V with 2,4,6-triaminopyrimidine (IV) in water atreflux gave 2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxyaldehyde (III;Y═CHO). The structure of this compound was confirmed as describedhereinafter in Example 8B by the alkaline potassium permanganateoxidation of the formyl group and hydrolysis of the 4-amino group togive the known 2-amino-4(3H)oxopyrido[2,3-d]pyrimidine-6-carboxylic acid(VIII) [R. Bernetti, F. Mancini and C. C. Price, J. Org. Chem., 27, 2863(1962); D. M. Mulvery, S. G. Cottis and H. Tieckelmann, J. Org. Chem.,29, 2903 (1964)]. An authentic sample of VIII was prepared as describedhereinafter in Example 8A by alkaline potassium permanganate oxidationof 2-amino-6-methyl-4(3H)oxopyrido[2,3-d]pyrimidine, which wassynthesized by the method of E. Stark and E. Breitmaier, Tetrahedron,29, 2209 (1973). It has been established that in the2,4-diaminopyrido[2,3-d]pyrimidine ring system, the 4-amino functionundergoes alkaline hydrolysis readily [R. Tratner, G. Elion, G.Hitchings, and D. Sharefkin, J. Org. Chem., 29, 2674 (1964)].

Although the mechanism of the condensation reaction is unknown, two ofthe formyl groups or potential formyl groups of V must react with theenamine moiety of the 4-aminopyrimidine with the elimination of eitherwater or dimethylamine. The initial reaction involves the electrophilicattack of one formyl group or derivative either with the 5-position ofthe pyrimidine ring or with the 4-amino group to give a Schiff basefollowed by cyclization of the resulting monocyclic intermediate to givethe desired bicyclic ring system. In the J. Org. Chem. reference above,Price et al observed that pyrido[2,3-d]pyrimidines were readily formedunder mild conditions from 4-aminopyrimidines and malondialdehydescontaining electron-withdrawing groups. Compound V can be considered amalondialdehyde derivative substituted with an electron-withdrawinggroup.

The compound of Formula III (Y=CH₂ OH) is prepared by reducing thecompound of Formula III (Y=CHO) in N,N-dimethylacetamide (DMAC) withsodium borohydride.

The compound of Formula III (Y=CH₂ Br) is prepared by treating thecompound of Formula III (Y=CH₂ OH) with dibromotriphenylphosphorane inDMAC [procedure of J. R. Piper and J. A. Montgomery, J. Org. Chem., 42,208 (1977)]. This results in the production of the corresponding2,4-bis[(triphenylphosphoranylidene)amino] derivative, i.e., thecompound of Formula IIIA. The compound of Formula IIIA can be convertedto the compound of formula III (Y=CH₂ Br) by the procedure described inthe cited article by Piper and Montgomery. Either the compound ofFormula IIIA or the compound of Formula III (Y=CH₂ Br) can be used toreact with a dialkyl p-aminobenzoyl-L-glutamate (VI); although it ispreferred to use the compound of Formula IIIA, as illustrated in Example3, Part B, which is formed in situ.

The compound of Formula IIIB (Z=CH₂ OH), i.e.,2-amino-6-(hydroxymethyl)-4(3H)oxopyrido[2,3-d]pyrimidine, can beprepared by hydrolyzing the 4-amino group of the compound of Formula III(Y=CH₂ OH) according to the method described in the earlier citedpublication by R. Tratner, G. Elion, G. Hitchings and D. Sharefkin.Conversion of the compound of Formula IIIB (Z=CH₂ OH) to2-amino-6-(bromomethyl)-4(3H)oxopyrido[2,3-d]pyrimidine (IIIB; Z=CH₂ Br)is accomplished by treatment with phosphorus tribromide according to theprocedure of S. Srinivasan and A. D. Broom, J. Org. Chem., 46, 1777(1981). In addition, the compound of Formula IIIB (Z=CH₂ Br) can beprepared by hydrolysis of the 4-amino group of the compound of FormulaIII (Y=CH₂ Br) with 48% hydrobromic acid according to the procedure ofJ. A. Montgomery, J. D. Rose, C. Temple, Jr. and J. R. Piper, "Chemistryand Biology of Pteridines", W. Pfleiderer, ed., Walter de Grugter,Berlin, 1976, p. 485.

The dialkyl p-(substituted amino)benzoyl-L-glutamates (VI) are preparedas described by A. H. Calvert, T. R. Jones, A. L. Jackman, S. J. Brownand K. R. Harrap, in "Advances in Tumour Prevention, Detection andCharacterization, Vol. 5: Human Cancer. Its characterization andTreatment", W. Davis, K. R. Harrap and G. Stathopoulos, eds., ExcerptaMedica, Amsterdam, 1980, P. 272; and T. R. Jones, A. H. Calvert, A. L.Jackman, S. J. Brown, M. Jones and K. R. Harrap, Europ. J. Cancer, 17,11 (1981).

Reductive alkylation of diethyl p-aminobenzoyl-L-glutamate) (VI; R=H; R₁=C₂ H₅) with III (Y=CHO) and hydrogen in 70% acetic acid containingRaney nickel gave a 32% yield of 5-deazaaminopterin diethyl ester.Saponification of the ester groups in a mixture of dimethylsulfoxide-water at ambient temperature gave an 87% yield of5-deazaaminopterin (II, R=H). Methylation of the latter compound wasaccomplished by treatment of II (R=H) with formaldehyde and sodiumcyanoborohydride in aqueous solution at pH 6.4 to give an 85% yield of5-deazamethotrexate, (II, R=CH₃). The structure of II (R=CH₃) wasestablished as described hereinafter in Example 8C by oxidation withalkaline potassium permanganate to give the previously prepared2-amino-4(3H)oxopyrido[2,3-d]pyrimidine-6-carboxylic acid (VIII), whichindicated that methylation had occurred either on the 4- or 10-aminogroup. Methylation of the 4-amino group was eliminated fromconsideration by alkaline hydrolysis of the 4-amino group to give5-deaza-10-methylfolic acid (I, R=CH₃).

The preferred route for the preparation of I (R=H) involves thehydrolysis of 5-deazaaminopterin diethyl ester (VII; R=H; R₁ =C₂ H₅) inaqueous sodium hydroxide at reflux temperature, which resulted inreplacement of the 4-amino group as well as hydrolysis of the esterfunctions to give a 79% yield of 5-deazafolic acid (I, R=H). Methylationof the compound of Formula I (R=H) with formaldehyde and sodiumcyanoborohydride gave an 84% yield of 5-deaza-10-methylfolic acid (I,R=CH₃), which was identical to that prepared by the alkaline hydrolysisof the compound of Formula II (R=CH₃). The structures of I (R=H andCH₃), II (R=H and CH₃), and 5-deazaaminopterin diethyl ester wereconfirmed by elemental analysis, ¹ H-NMR and mass spectral data.

Reaction of the compound of Formula IIIB (Z=CH₂ Br) with diethylp-aminobenzoyl glutamate (VI; R=H; R₁ =C₂ H₅) gaveN-[4-[(2-amino-4(3H)oxopyrido[2,3-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glutamate(VIIA; R=H; R₁ =C₂ H₅), which was converted to a compound of Formula I(R=H) by saponification.

Compounds of Formulas I and II form pharmaceutically acceptable saltswith both organic and inorganic acids. Examples of suitable acids forsalt formation are hydrochloric, sulfuric, phosphoric, acetic, citric,oxalic, malonic, salicyclic, malic, fumaric, succinic, ascorbic, maleic,methanesulfonic, and the like. The salts are prepared by contacting thefree base form with an equivalent amount of the desired acid in theconventional manner. The free base forms may be regenerated by treatingthe salt form with a base. For example, dilute aqueous base solutionsmay be utilized. Dilute aqueous sodium hydroxide, potassium carbonate,ammonia, and sodium bicarbonate solutions are suitable for this purpose.The free base forms differ from their respective salts forms somewhat incertain physical properties such as solubility in polar solvents, butthe salts are otherwise equivalent to their respective free base formsfor purposes of the invention.

Therapeutic compositions containing compounds of Formulas I and II areuseful for ameliorating cancer diseases in mammals. The activeingredients of the therapeutic compositions inhibit transplanted mousetumor growth when administered in amounts ranging from about 5 mg toabout 200 mg per kilogram of body weight per day. A preferred dosageregimen for optimum results would be from about 5 mg to about 50 mg perkilogram of body weight per day, and such dosage units are employed thata total of from about 350 mg to about 3.5 grams of the active compoundfor a subject of about 70 kg of body weight are administered in a24-hour period. This dosage regimen may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. A decidedpractical advantage is that the active compound may be administered inany convenient manner such as by the oral, intravenous, intramuscular orsubcutaneous routes.

The active compounds may be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or they may beenclosed in hard or soft shell gelatin capsules, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 and about 60% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 5 and about 200 milligrams of active compound.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen or cherry flavoring. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparations and formulations.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compound as a free base orpharmaceutically acceptable salt can be prepared in water suitably mixedwith a surfactant such as hydroxypropylcellulose. Dispersions can alsobe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuitable as unitary dosages for the mammalian subjects to be treated;each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the dosageunit forms are dictated by and directly dependent on (a) the uniquecharacteristics of the active material and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active material for the treatment of disease inliving subjects having a diseased condition in which bodily health isimpaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically-acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from about 0.1 to about 400 mg, withfrom about one to about 30 mg being preferred. Expressed in proportions,the active compound is generally present in from about 0.1 to about 400mg/ml of carrier. In the case of compositions containing supplementaryactive ingredients, the dosages are determined by reference to the usualdose and manner of administration of the said ingredients.

The following examples illustrate the best modes known for carrying outthis invention:

EXAMPLE 1 2,4-Diamino-6-(carboxaldehyde)pyrido[2,3-d]pyrimidine (III;Y=CHO)

Phosphorus oxychloride (27.5 ml, 46.0 g, 300 mmol) was added over 15minutes with stirring to N,N-dimethylformamide (11.0 g, 150 mmol), whichwas cooled with an ice bath. After stirring at room temperature for 1hour, the reaction mixture was treated with bromoacetic acid (13.9 g,100 mmol). The resulting solution, protected by a calcium chloride tubewas heated at 92° C. for 10 hours and evaporated to dryness in vacuo.The colored oil (˜30 g) was dissolved in water (1000 ml), and thesolution was neutralized with 50% sodium hydroxide to pH 7. Afteraddition of 2,4,6-triaminopyrimidine (5.00 g, 40.0 mmol), the solutionwas refluxed for 3 hours and filtered hot through a fluted filter. Thefiltrate was cooled and the solid that precipitated was collected byfiltration and dried in vacuo over P₂ O₅ : yield, 2.53 g (33%). Massspectrum, m/e 189 (M⁺). HPLC [0.1M NH₄ OAc (pH 3.6)--CH₃ OH (9:1)]indicated that this product was 86% pure. A sample (200 mg) wasdissolved in 0.1N HCl (15 ml) and diluted with acetone (225 ml) toprecipitate impure III (Y=CHO): yield, 91 mg. The filtrate wasevaporated to dryness under reduced pressure and the residue was driedin vacuo over P₂ O₅ to give Compound III (Y=CHO): yield, 128 mg; mp,gradual darkening and decomposition with white sublimate when taken to360° C. λ_(max) nm (ε×10⁻³): 0.1N HCl-258 (16.4), 317 (9.12), 326 sh(8.24); pH 7-263 (15.0), 316 (10.1), 345 (10.8); 0.1N NaOH-254 (13.2),267 (13.5), 316 (8.56), 347 (10.0). ¹ H-NMR (CF₃ CO₂ D, 6% w/v), 9.48 s,9.75 s (5-CH, 7-CH), 10.21 s (6-CHO).

Anal. Calcd for C₈ H₇ N₅ O.HCL.1.3H₂ O: C, 38.57; H, 4.30; N, 28.12.Found: C, 38.44; H, 4.15; N, 28.14.

EXAMPLE 2 2,4-Diamino-6-(hydroxymethyl)pyrido[2,3-d]pyrimidine(III;Y=CH₂ OH)

A solution of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxaldehydehydrochloride (200 mg, 0.884 mmol) in DMAC (40 ml) at 0° C. was treatedwith 1N NaOH (0.884 ml, 0.884 mmol) followed by a suspension of NaBH₄(16.7 mg, 0.442 mmol) in DMAC (1 ml). The solution was stirred at 25° C.for 30 minutes and evaporated to dryness under high vacuum. A stirredsuspension of the residue in H₂ O (5 ml) was adjusted to pH 3 with 6NHCl, stirred for 5 minutes and adjusted to pH 7 with 1N NaOH. Theprecipitate was collected by filtration, washed with H₂ O and dried invacuo (P₂ O₅); yield 121 mg (51%); mass spectrum, m/e 191 (M)⁺, 87 (M)⁺for DMAC, 36 (M)⁺ for HCl; λ_(max) nm (ε×10⁻³): 0.1N HCl-274 (6.91), 318(8.21), 330 sh (7.17), 362 (1.77); pH 7-248 (18.1), 271 (9.85), 338(6.52); 0.1N NaOH-248 (19.7), 271 (10.3), 345 (7.14).

Anal. Calcd for C₈ H₉ N₅ O.1.1HCl.0.4(CH₃)₂ --NCOCH₃ : C, 43.32; H,5.19; N, 28.42. Found: C, 43.48; H, 5.24; N, 28.48.

EXAMPLE 3 DiethylN-[4-[(2,4-diaminopyrido[2,3-d]pyrimidine-6-yl)methylamino]benzoyl]-L-glutamate(VII; R=H; R₁ =C₂ H₅)

A. A solution of Compound III (Y=CHO) (1.47 g, 5.90 mmol) in warm 70%acetic acid (59 ml) was cooled to 25° C., treated with diethylp-aminobenzoyl-L-glutamate (2.28 g, 7.08 mmol) and hydrogenated in thepresence of Raney nickel (6.3 g, weighed wet) at 25° C. and atmosphericpressure for 17 hours. The mixture was filtered and the catalyst waswashed with 70% acetic acid (25 ml). The combined filtrate and wash wasevaporated to dryness under high vacuum, and a solution of the residuein ethanol was filtered into 2N Na₂ CO₃ (60 ml). The mixture was stirredto give a homogeneous powder which was collected, washed with water anddried. A solution of the resultant powder in boiling ethanol (415 ml)was filtered hot and evaporated to dryness in vacuo. The residue wastriturated with CHCl₃ (85 ml), collected by filtration and the solid waswashed with additional CHCl₃ (40 ml). A suspension of the solid inboiling ethanol (140 ml) was stirred for 20 minutes and refrigerated.The product was collected by filtration and dried in vacuo (P₂ O₅):yield 945 mg (32%), mp 262° C. (Kofler Heizbank). Mass spectrum, m/e 496(M+1)⁺ ; λ_(max) nm (ε×10⁻³): 0.1N HCl-218 (42.4), 280 sh (19.3), 300(22.0); pH 7-218 (36.4), 249 (20.2), 280 sh (22.3) 297 (23.6), 355 sh(6.10); 0.1N NaOH-249 (22.0), 280 (23.8), 297 sh (22.5), 345 (7.23). ¹H-NMR(DMSO-d₆, 6% w/v), δ1.18 m CH₃, 2.05 m (CH₂ CH₂ CO), 2.43 t (CH₂CO), 4.08 m (CH₂ O), 4.32 m (CH₂ N, CHN), 6.31 s, 7.51 s (NH₂), 6.67 d,7.69 d (C₆ H₄), 6.71 s (CH₂ NH), 8.25 d (CONH), 8.41 d (5-CH, J=2.0 Hz),8.66 d (7-CH, J=2.0 Hz).

Anal. Calcd for C₂₄ H₂₉ N₇ O₅ : C, 58.17; H, 5.90; N, 19.79. Found: C,57.91; H, 6.24; N, 19.55.

Evaporation of the filtrate and trituration of the residue with ethanolgave an additional 123 mg of less pure product, mp 246° C.

B. A solution of triphenylphosphine (430 mg, 1.64 mmol) in anhydrousDMAC (4 ml) at 0° C. was treated dropwise under N₂ with bromine (84 μL,1.64 mmol). The solution was stirred at 0° C. for 15 minutes, treatedwith the compound obtained in Example 2 (100 mg, 0.76 mmol), and stirredat 25° C. for 1.7 hours to give the compound of Formula IIIA. Thissolution was treated with diethyl p-aminobenzoyl-L-glutamate (194 mg,0.603 mmol), stirred at 25° C. for 17 hours and poured into ice water(40 ml). The resulting solution (pH 2) was filtered to removetriphenylphosphine oxide, adjusted to pH 6.8 with 50% NaOH, and cooledin an ice bath. The precipitate of crude product (VII; R=H, R₁ =C₂ H₅)was collected by centrifugation, washed with H₂ O and dried in vacuo (P₂O₅); yield 136 mg (73%). The retention time (HPLC) and rf value (TLC) ofthis product were identical to those obtained from the analytical sampleobtained in Part A of this example. Mass spectrum: m/e 496 (M+1)⁺.

EXAMPLE 4N-[4-[(2-Amino-4(3H)-oxopyrido[2,3-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glutamicAcid (I, R=H)

A suspension of the product obtained in the previous example, VII (R=H;R₁ =C₂ H₅) (100 mg, 0.202 mmol) in O₂ free 1N NaOH (4 ml) was stirred atreflux temperature under N₂ for 4.25 hours and acidified to pH 3.1 with6N HCl. The precipitate was collected by filtration and dried in vacuo.A solution of the solid in 1N HCl (0.5 ml) was diluted with water (0.5ml), filtered, diluted with water (9 ml) and adjusted to pH 3.1 with 1NNaOH. The precipitate was collected by filtration, washed with water atpH 3.1 and dried in vacuo (P₂ O₅): yield 74 mg (79%), mp indefinite;mass spectrum, m/e 441 (M+1)⁺ ; λ_(max) nm (ε×10⁻³): 0.1N HCl-213(37.0), 280 (23.9), 297 sh (20.6), 350 (7.35); pH 7-216 (40.8), 278(24.9), 295 sh (23.8); 0.1N NaOH-243 (22.9), 278 (24.0), 295 sh (22.7),345 sh (7.58); ¹ H-NMR (CF₃ CO₂ D, <6% w/v), 2.56 (CH₂ CH₂ CO), 2.82 t(CH₂ O), 5.11 m (CHN, CH₂ N), 7.87 d, 8.15 d (C₆ H₅), 8.98 s, 9.10 s(5-CH, 7-CH).

Anal. Calcd for C₂₀ H₂₀ N₆ O₆.1.1H₂ O: C, 52.20; H, 4.86; N, 18.26.Found: C, 52.00; H, 4.92; N, 18.54.

EXAMPLE 5N-[4-[(2,4-Diaminopyrido[2,3-d]pyrimidine-6-yl)methylamino]benzoyl]-L-glutamicacid (II, R=H)

A solution of the product obtained in Example 3, VII (R=H; R₁ =C₂ H₅)(359 mg, 0.724 mmol) in dimethyl sulfoxide (10 ml) under N₂ was treatedwith 1N NaOH (1.81 ml, 1.81 mmol), stirred in a stoppered flask under N₂for 6 hours, and evaporated to dryness in vacuo at <30° C. A solution ofthe residue in water (18 ml) was filtered and acidified to pH 3.6 with1N HCl. The precipitate was collected by filtration, washed with waterat pH 3.6 and dried in vacuo (P₂ O₅); yield 297 mg (87%), mp indefinite(softens above 200° C.); mass spectrum, m/e 440 (M+1)⁺ ; λ_(max) nm(ε×10⁻³): 0.1N HCl-218 (40.5), 280 sh (16.9), 300 (18.8); pH 7-218(38.5), 245 (19.2), 280 (23.9), 296 sh (22.7); 0.1N NaOH-248 (22.0), 280(24.4), 296 sh (22.7), 345 (7.75); ¹ H-NMR (DMSO-d₆, 6% w/v), δ2.00 m(CH₂ CH₂ CO), 2.29 t (CH₂ CO), 4.36 m (CHN, CH₂ N), 6.66 d, 7.68 d (C₆H₄), 7.41 (NH₂), 8.04 m (NH₂, NH, CO₂ H), 8.52 d, 8.70 d (5-CH, 7-CH).

Anal. Calcd. for C₂₀ H₂₁ N₇ O₅.1.9H₂ O: C, 50.72; H, 5.28; N, 20.70.Found: C, 50.86; H, 5.43; N, 20.50.

EXAMPLE 6N-[4-[[(2,4-Diaminopyrido[2,3-d]pyrimidine-6-yl)methyl]methylamino]benzoyl]-L-glutamicacid (II, R=CH₃)

A suspension of Compound II (R=H) (100 mg, 0.211 mmol) in O₂ free water(5 ml) under N₂ was adjusted to pH 6.4 with 1N NaOH to give a solutionwhich was treated with 38% HCHO (83.1 μl, 1.14 mmol) followed by NaBH₃CN (19.9 mg, 0.317 mmol). The solution was maintained at pH 6.4 bygradual addition of 1N HCl over a period of 45 minutes. The solution wasstirred under N₂ for 23 hours, filtered and acidified to pH 3.6 with 1NHCl. The product was collected by filtration, washed with water at pH3.6 and dried in vacuo (P₂ O₅): yield 97 mg (94%), mp indefinite(softens and darkens above 217° C.); mass spectrum, m/e 454 (M+1)⁺ ;λ_(max) nm (ε×10⁻³): 0.1N HCl-221 (37.1), 311 (19.0); pH 7-219 (35.1),247 (18.1), 305 (25.2); 0.1N NaOH-249 (19.9), 305 (25.0), 355 sh (6.15;¹ H-NMR (DMSO-d₆, <5% w/v), δ2.00 m (CH₂ CH₂ CO), 2.28 t (CH₂ CO), 3.12s (CH₃), 4.32 m (CHN), 4.66 s (CH₂ N), 6.78 d, 7.72 d (C₆ H₄), 8.31 d(5-CH), 8.59 d (7-CH).

Anal. Calcd. for C₂₁ H₂₃ N₉ O₅.2H₂ O: C, 51.53; H, 5.56; N, 20.03.Found: C, 51.54; H, 5.47; N, 20.35.

EXAMPLE 7N-[4-[[(2-Amino-4(3H)-oxopyrido[2,3-d]pyrimidine-6-yl)methyl]methylamino]benzoyl]-L-glutamicacid (I, R=CH₃)

A. A suspension of I (R=H) (60 mg, 0.13 mmol) was methylated by theprocedure used for the preparation of II (R=CH₃). The reaction solutionafter filtration was diluted with oxygen free water (3 ml) and acidifiedto pH 3.1 with 1N HCl. The product was collected, washed with water atpH 3.1 and dried in vacuo (P₂ O₅); yield 53 mg (84%), mp indefinite;mass spectrum, m/e 455 (M+1)⁺ ; λ_(max) nm (ε×10⁻³): 0.1N HCl-215(35.1), 280 (19.0), 306 (20.8), 355 sh (6.85); pH 7-216 (38.0), 274(19.0), 306 (27.3); 0.1N NaOH-242 (22.9), 275 sh (17.4), 307 (25.4); ¹H-NMR (DMSO-d₆, 5% w/v), δ2.02 m (CH₂ CH₂ CO₂ H), 2.35 t (CH₂ CO), 3.09s (CH₃), 4.37 m (CHN), 4.73 s (CH₂ N), 6.82 d, 7.75 d (C₆ H₄), 8.03 d(5-CH), 8.19 d (NH), 8.55 d (7-CH).

Anal. Calcd. for C₂₁ H₂₂ N₆ O₅.H₂ O.0.75HCl: C, 52.14; H, 5.16; N,17.37. Found: C, 52.12; H, 5.12; N, 17.47.

B. A solution of Compound II (R=CH₃) (50 mg) was hydrolyzed by theprocedure of Example 4 for the preparation of Compound I (R=H) to give a64% yield of Compound I (R=CH₃). HPLC and uv data indicated that thisproduct was identical to that prepared in A above.

EXAMPLE 8 2-Amino-4(3H)-oxopyrido[2,3-d]pyrimidine-6-carboxylic Acid(VIII)

A. To a solution of 2-amino-6-methyl-4(3H)-oxopyrido[2,3-d]pyrimidine(177 mg, 1.00 mmol) in 1N NaOH (60 ml) at reflux temperature was addedwith stirring an aqueous solution of 0.2M potassium permanganate over aperiod of about 1 hour. After the excess permanganate was destroyed withsodium bisulfite, the resulting hot mixture was filtered through Celite.The filtrate was adjusted to ˜pH 3 with HCl and allowed to stand at roomtemperature for 18 hours. The solid that precipitated (170 mg) wascollected by filtration, dissolved in 2N NaOH, and the solution wascooled to deposit the sodium salt of the product. The salt was collectedby filtration, dissolved in water, and the solution was adjusted to pH2-3 with HCl. The solid that deposited was collected by filtration anddried in vacuo over P₂ O₅ : yield, 67 mg (29%); mp 265° C. HPLC [0.1MNa₂ HPO₄ (pH 7)-CH₃ CN (92:8)] showed that this sample was homogeneous.λ_(max) nm (ε×10⁻³): 0.1N HCl-216 (35.9), 266 (14.5), 306 (6.70), 315 sh(5.35); pH 7-216 (26.8), 232 sh (17.8), 283 (11.4), 310 sh (5.93), 321sh (5.37); 0.1N NaOH-246 (22.8), 292 (10.1), 332 (7.20). ¹ H-NMR (NaOD,5% w/v), δ8.76 d (7-CH, J=1.5 Hz), 9.06 d (5-CH).

Anal. Calcd for C₈ H₆ N₄ O₃.0.6HCl: C, 42.13; H, 2.92; N, 24.57. Found:C, 42.04; H, 2.80; N, 24.41.

B. Treatment of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxaldehyde(III; Y=CHO) (186 mg, ˜0.980 mmol) by the procedure described in Aresulted in hydrolysis of the 4-amino group and oxidation of the formylgroup to give VIII: yield, 158 mg; Field desorption mass spectrum, m/e206 (M⁺). The HPLC chromatogram (co-injection) of this product wasidentical with that of Compound VIII prepared in A.

C. A solution ofN-[4-[[(2,4-diaminopyrido[2,3-d]pyrimidin-6-yl)methyl]methylamino]benzoyl]-L-glutamicacid (II, R=CH₃) (5.00 mg, 0.010 mmol) in 2 ml of 1N NaOH was treatedwith KMnO₄ (1.62 mg, 0.010 mmol), heated at 95° C. for 3 hours, filteredand adjusted to pH 8 with 1N HCl. An HPLC chromatogram indicated thepresence of VIII (˜22% yield) and unreacted II (R=CH₃) (˜50% recovery).The ultraviolet spectrum (240-360 nm) of the eluted Compound VIII wasidentical to the ultraviolet spectrum of an authentic sample.

Cell culture cytotoxicity data and activity against lymphocytic leukemiaP388 in mice for Compounds I, II and VII (R=H; R₁ =C₂ H₅) are set forthin Table 1.

                  TABLE 1    ______________________________________    Cell Culture Cytotoxicity Data.sup.a and Activity    Against Lymphocytic Leukemia P388 in Mice.sup.b                        P388.sup.d    Compound        ED.sub.50 μM.sup.c                              dose, mg/kg                                         % ILS    ______________________________________    I(R = H)        6.1       100        17    I(R = CH.sub.3) 11.2      200        11    II(R = H)       0.013     --         --    II(R = CH.sub.3)                    0.004      4          92.sup.e    VII(R = H; R.sub.1 = C.sub.2 H.sub.5)                    0.052      1         118    Methotrexate    0.001      2          61.sup.f    ______________________________________     .sup.a Human epidermoid carcinoma cell No. 2. R. I. Geran, N. H.     Greenberg, M. M. MacDonald, A. M. Schumacher, and B. J. Abbott, Cancer     Chemotherapy Reports, Part 3, Vol. 3 (No. 2), 1971.     .sup.b Reference in a.     .sup.c Concentrations inhibiting colony formation by 50% after 12 days as     determined in plastic flask. L. L. Bennett, Jr., H. D. Schnetti, N. H.     Vail, P. W. Allan, and J. A. Montgomery, Mol. Pharmacol., 2, 432 (1966).     .sup.d CDF.sub.1 mice inoculated with 10.sup.6 P388 cells     intraperitoneally; drug administered intraperitoneally on qd 1-5 days.     .sup.e One 30thday survivor.     .sup.f Administered intraperitoneally on qd 1-9 days.

What is claimed is:
 1. Intermediates useful in the production of 5-deazaanalogs of folic acid, N¹⁰ -substituted folic acid, aminopterin, and N¹⁰-substituted aminopterin consisting of compounds having the structure:##STR13## wherein Y is CH₂ OH or CH₂ Br. 2.2,4-Diamino-6-(hydroxymethyl)pyrido[2,3-d]pyrimidine.